The objective of this proposal is twofold, to develop an in vitro co-culture system in which the interaction between two different cell types can be studied, and to evaluate the role of the glial cell in the differentiation and maintenance of the Blood-Brain Barrier (BBB) capillary endothelium of the brain. This project will utilize a mouse freeze-injury model to study these processes in the regenerating cerebral microvessell in vivo, and a co-culture sytem where the interaction between glial and endothelial cells can be studied under controlled in vitro conditions. The specific aims in vivo are to thoroughly investigate the temporal sequence and relationships of endothelial tight junction formation, early appearance of glial foot processes of the bascular basement membrane, the completion of the glial sheath around the regenerating vessel, restoration of barrier function to tracers, the fate of endothelial cell Factor VIII antigen during regeneration, and induction of gamma-glutamyl transpeptidase (Gamma-GTP) in the new endothelium. In vitro, the aims are to determine the mechanisms by which endothelial cells acquire Gamma-GTP in the presence of glial cells in co-culture, to test the glial cell's ability to induce other differentiated endothelial characteristics, and to delineate the extent to which glial cells can induce Gamma-GTP in endothelium from extracranial sites. The accomplishment of these aims will add significantly to our understanding of the microvascular regeneration process following a variety of clincal and pathological conditions where alterations in vascular integrity have resulted in varying degrees of brain damage. For example, arteriolar sclerosis with medial fibrosis and hyalinization is a frequent accompaniment to the small stroke syndrome and lacunar infarcts, while arteriolar necrosis will microaneurysm formation accopanies hypertensive hemorrhage. In addition, infarcts, trauma, anoxia, neoplasms and infection all affect the integrity of the microvasculature. Of equal importance will be the potential generalization of these or similar co-culture techniques to other cell-cell interaction studies in the biomedical field.